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I use the next versions:

gatk   4.4.0.0
minimap2   2.26-r1175
samtools   1.19

I have a fastq file. And try to implement gatk MergeBamAlignment to generate a new bam-file:

java -jar gatk.jar FastqToSam --FASTQ .fastq --OUTPUT u.bam
--SAMPLE_NAME sample1 -RG readgroup -PL ILLUMINA
minimap2 -a hg38.fa .fastq -o A.sam
java -jar gatk.jar SortSam -I A.sam -O A.sort.bam -SO queryname
java -jar gatk.jar SortSam -I u.bam -O u.sort.bam -SO queryname
java -jar gatk.jar MergeBamAlignment -O m.bam -R hg38.fa -UNMAPPED_BAM u.bam -ALIGNED_BAM A.sort.bam

But I receive error on the output of MergeBamAlignment:

INFO    2023-12-21 19:20:41     SamAlignmentMerger      Processing SAM file(s): [./myprogs/data/NR.Aligned.sort.bam] 

[Thu Dec 21 19:20:42 2023] picard.sam.MergeBamAlignment done. 

Elapsed time: 0.02 minutes.

Runtime.totalMemory()=486539264 

To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp

java.lang.ArrayIndexOutOfBoundsException: Index 86 out of bounds for length 86
at htsjdk.samtools.util.SequenceUtil.calculateMdAndNmTags(SequenceUtil.java:990)
at picard.sam.AbstractAlignmentMerger.fixNmMdAndUq(AbstractAlignmentMerger.java:611)
at picard.sam.AbstractAlignmentMerger.mergeAlignment(AbstractAlignmentMerger.java:586)
at picard.sam.SamAlignmentMerger.mergeAlignment(SamAlignmentMerger.java:181)
at picard.sam.MergeBamAlignment.doWork(MergeBamAlignment.java:368)
at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:289)
at org.broadinstitute.hellbender.cmdline.PicardCommandLineProgramExecutor.instanceMain(PicardCommandLineProgramExecutor.java:37)
at org.broadinstitute.hellbender.Main.runCommandLineProgram(Main.java:160)
at org.broadinstitute.hellbender.Main.mainEntry(Main.java:203)
at org.broadinstitute.hellbender.Main.main(Main.java:289)

I have diagnosed u.sort.bam and A.sam.bam with ValidateSamFile:

java -jar gatk.jar ValidateSamFile -I u.sort.bam -M SUMMARY -R hg38.fa
No errors found
[Thu Dec 21 00:32:34 2023] picard.sam.ValidateSamFile done. Elapsed time: 0.00 minutes. 
Runtime.totalMemory()=234881024 

Tool returned: 
0 

java -jar gatk.jar ValidateSamFile -I A.sort.bam -M SUMMARY -R hg38.fa
ERROR    2023-12-21 01:17:28    ValidateSamFile    Requesting earlier reference sequence: 23 < 49 
[Thu Dec 21 01:17:28 2023] picard.sam.ValidateSamFile done. Elapsed time: 0.18 minutes.
Runtime.totalMemory()=2113929216
To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp  

Tool returned:
-1

I have done the same actions only with --MD in minimap2. But the same errors occur. I would like to find out how to fix this problem. I use the latest versions of the programs. I have downloaded hg38.fa each one time, but it didn't change the situation. I acts in according with gatk's article 'Data pre-processing for variant discovery' : https://gatk.broadinstitute.org/hc/en-us/articles/360035535912-Data-pre-processing-for-variant-discovery

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  • $\begingroup$ I don't think that you need to resort as minimap should output the reads in the same order that they were ingested $\endgroup$
    – Yossi Farjoun
    Commented Dec 30, 2023 at 15:31
  • $\begingroup$ However, the fact that the output of gatk SortSam isn't valid is disconcerting. Can you see if it's a small part of your fasta file that is the problem? perhaps chop it in half and see if you can reproduce it on a smaller fastq? $\endgroup$
    – Yossi Farjoun
    Commented Dec 30, 2023 at 15:36

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